Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host’s roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial 59Fe2+ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for 52Mn2+ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.


Introduction
Light has been a vital driver for life on Earth, but it has also proved to be threatening to many organisms due to the photodynamic effect [1]. Over time, organisms have developed adaptations to cope with, or even to exploit, light. It is common knowledge that, as autotrophs, light has vital importance to the growth and development of higher plants. What is less known is how light might impact the belowground processes involving the roots and/or the microorganisms that colonize them. In a recent study, it was demonstrated

Bacteria Growth
Three functional mutant strains of Azospirillum brasilense (HM053, ipdC, and FP10) were used in the study and obtained through a material transfer agreement between the Federal University of Paraña (UFPR, Curitiba, PR 174CEP 81531-980, Brazil) and the corresponding author's institution. The HM053 mutant is a natural mutant of the wild-type strain of A. brasilense FP2 (Sp7 ATCC 29145 Nif + Sm r Nal r ) screened through its resistance to ethylenediamine (EDA r ) [25,26]. For the ipdC mutant, the indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of auxin (indole-3-acetic acid) biosynthesis in Azospirillum brasilense, was functionally disrupted in a site-specific manner using a SacB-cassette insertion into the ipdC gene of wild-type FP2 (Sp7 ATCC 29145) followed by homologous recombination. The method allowed for the construction of the ipdC mutation strain without unwanted sequence changes and relied on the λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) which works well for generating single point mutations, small insertions or replacements, as well as deletions of any size, in a bacterial gene [27]. The resultant knock-out strain exhibited a significant reduction in auxin biosynthesis to a level of 10% that of the wild-type strain [19]. The FP10 mutant was obtained by N-nitrosoguanidine mutagenesis of the FP2 wild-type strain of A. brasilense and isolated by growth on glutamate medium [28].
The functional mutants were grown in liquid NFbHP-lactate medium at pH 6.5 following published procedures [16]. The medium contained 20 mM ammonium chloride (NH 4 Cl) as a nitrogen source and streptomycin antibiotic (80 µg mL −1 ). The cultures were grown in a shaking incubator at 30 • C and 130 rpm in 10 mL volume, then re-propagated into 40 mL volume approximately 24 h prior to starting the tracer administration. The bacteria were grown under two treatment types: 24 h light and 24 h dark. During the light experiments, bacteria were propagated directly under a 30 cm × 30 cm red-blue LED light panel intended for indoor plant growth (200 µmol m −2 s −1 total intensity) that was positioned over the shaking incubator. Lights were left on for the entirety of the bacteria growth period. Dark experiments involved propagation of the bacteria in the dark, handled under indirect light, and grown in the shaking incubator at 30 • C while shielded from any ambient light.

Plant Growth
Maize kernels (Hybrid 8100) from Elk Mound Seed Co. (Elk Mound, WI, USA) were dark germinated at room temperature for two days on sterilized paper towels wetted with sterile water in a petri dish. Seedling's roots were inoculated with isolated bacteria strains suspended in sterile water for 3 h prior to transplanting to a growth pouch wetted with sterile Hoagland's basal salt solution for approximately one week. They were then transferred to eight-inch plastic cones filled with Turface™ expanded clay matrix (Profile Products, LLC., Buffalo Grove, IL, USA) where the bottom portion of the cone was immersed in de-ionized water. Nutrients were introduced as Hoagland's solution every three days. Growth conditions consisted of 12 h photoperiods, 500 µmol m −2 s −1 light intensity, and temperatures of 25 • C/20 • C (light/dark) with humidity at 70-80% for two weeks. For BNF studies, roots were removed from the Turface™ and subjected to the acetylene reduction assay described below for measuring microbial nitrogenase activity. A subset of each root mass collected was also subjected to drop plate analysis to measure the extent of microbial root inoculation.

Acetylene Reduction Assay (ARA)
ARAs were conducted to examine functionality of the nitrogenase enzyme under light and dark growth conditions. In this approach, acetylene gas is used to measure nitrogenase activity by its ability to reduce acetylene to ethylene. For details on the ARA, see the Supplemental Methods.
The bacteria were grown in association with maize roots, which were harvested, weighed (typically~1 gfw) and placed in a 500-mL Mason jar where the top was modified with a gas sampling port to introduce the acetylene gas and withdraw samples later for gas chromatography analysis ( Figure S2). For details on gas chromatography analysis, see the Supplemental Methods. Ethylene data from this analysis were normalized to CFUs per gram of root tissue as determined by Drop Plate Assays in each experiment. For details on the Drop Plate Assay, see the Supplemental Methods.
For light sensitivity studies, the Mason jars were exposed to full-spectrum white light using a white LED light panel (200 µmol m −2 s −1 intensity) or individual red LED or blue LED light panels (800 µmol m −2 s −1 intensities). For full darkness measurements, Mason jars were wrapped in aluminum foil. For consistency, all experimental measurements were carried out at 30 • C.

Microbial 59 Fe Uptake and Metabolic Transformation
Radioactive 59 Fe +3 and 59 Fe 2+ (t 1/2 = 44.5 days) were purchased in 1 mCi doses (equivalent to 37 MBq) from Perkin Elmer Life Sciences (Akron, OH, USA). Liquid cultures of bacteria were grown for 24 h in liquid NFbHP-lactate media prior to the start of a 59 Fe radiotracer study. On the day of an experiment, the cultures were centrifuged, washed with HyPure™ sterile deionized water (Cytiva HyClone Laboratory, Logan, UT, USA), and combined to yield a concentrated pellet of bacteria that was resuspended in 11 mL of HyPure™ water which had a slightly acidic pH of 5.5. A UV reading was taken at 600 nm (O.D. 600 ) using a 2 mL aliquot of the 11 mL sample. Of the 9 mL bacteria sample remaining, 9 aliquots of 1 mL were placed into 15 mL labeled Falcon tubes. Dosing with either 59 Fe 3+ or 59 Fe 2 + radiotracers as chloride salts was accomplished by adding radiometal diluted in HyPure™ sterile water to achieve 20 µCi doses (equivalent to 7.4 × 10 5 Bq) per 100 µL volume of water. Each dose possessed less than 1 nmole of nat .Fe carrier, bringing the concentration to 100 nM. Start times were recorded for radioactive decay correction back to common zero times. An aliquot of each diluted solution of 59 Fe radiotracer was counted in the NaI(Tl) gamma well counter and corrected for volume and detector efficiency back to a value for activity administered to each Falcon tube. Samples were incubated on a Unico TTR-2000 test tube rocker (United Products & Instruments, Inc., Dayton, NJ, USA) for 1, 3, and 5 h time points either under combined (red/blue) LED lights or shrouded in an aluminum foil blanket for dark studies. All radiotracer uptake data were normalized to a standard O.D. 600 value of 2.00.
At each time point, the appropriate samples were centrifuged, washed with sterile HyPure™ water three times, and re-suspended in 1 mL of 1 M HCl (pH << 1). An aliquot (100 µL) of each was removed for activity measurements in the NaI(Tl) gamma well counter for a measure of activity before extraction. The remainder of the washed bacteria were cell-disrupted for 2 min under 100% amplitude to burst open the cells for 59 Fe extraction. Once complete, samples were centrifuged for 2 min and the supernatant removed; 100 µL of the supernatant was removed for immediate counting in the NaI(Tl) gamma well detector, while the remaining 900 µL was used for iron oxidation state speciation via ion chromatography ( Figure S3). For details on the ion chromatography analysis, see the Supplemental Methods.
During ion chromatography, the 59 Fe 3+ and 59 Fe 2+ peaks were collected after separation and the amount of radioactivity in each fraction was measured with the NaI (Tl) gamma well detector. The percent tracer uptake was calculated for each bacterial functional mutant under light and dark conditions at 1, 3 and 5-h intervals. Additionally, the distribution of 59 Fe 3+ and 59 Fe 2+ ions present was measured to determine if transformation of the oxidation state of the tracer administered remained the same or was altered by our analytical workup of samples, or by the presence of the bacteria functional mutants. We note that while ferrous iron is soluble and relatively stable under anaerobic acidic conditions, under pH neutral aerobic conditions, ferrous iron can oxidize rapidly to its ferric form and be hydrolyzed to insoluble ferric hydroxide Fe(OH) 3 . Because our radiotracer uptake studies were conducted under mildly acidic conditions followed by strongly acidic conditions during analytical workup, we did not expect environmental oxidation of ferrous iron to be a problem. Even so, 59 Fe 2+ stability was examined without the presence of bacteria under our experimental protocol to verify this fact. Furthermore, prior studies on the hydrolysis of ferric chloride in dilute solutions showed that the rate at which ferric hydroxide is precipitated from such solutions decreases as the iron concentration decreases, reaching zero at a concentration of 2 nM [29]. This process is also pH-dependent and can be important at pH values above 6.5. As our studies were conducted in HyPure™ sterile water at pH 5.5, and at a concentration of 1 nM iron, it is unlikely that radiotracer hydrolysis and/or precipitation as ferric hydroxide was a factor to consider.

Luciferase Chemiluminescence ATP Assay
ATP levels were determined using BacTiter-Glo™ Microbial Cell Viability Assay Reagent (Promega, Madison, WI, USA). This was a luciferase-based assay and the ATP level was determined by measuring luminescence levels compared to ATP standards [30]. Measurement of ATP concentrations within the bacteria cells was performed using a published method of resuspending the bacteria pellet in 500 µL of deionized H 2 O and boiling the solution for 10 min to break down the cells [31]. For details on the experimental methods, see the Supplemental Methods. All data were normalized to a standard O.D. 600 value of 2.00.

Spectrophotometric Auxin Assay
Colorimetric analysis of auxin was performed using Salkowski reagent [32,33]. When carrying out the assay, 200 µL of bacterial culture was mixed with 300 µL of Salkowski reagent and kept in the dark for at least 30 min at room temperature. Additional samples from the same culture were centrifuged where the pellet was separated washed once, resuspended in 500 µL of H 2 O, and boiled for 10 min to release cellular auxin. Two hundred µL aliquot was mixed with 300 µL of Salkowski reagent and kept in the dark for at least 30 min at room temperature. Sample absorbances were measured at 536 nm using a spectrophotometer (Evolution 201 UV/VIS, ThermoFisher Scientific Inc., Waltham, MA, USA). For quantitative analysis, serial dilutions from 0 to 50 mg/L of auxin (Sigma Aldrich, St. Louis, MO, USA) were prepared and used as standards and the bacteria content in all analyses were normalized to a standard O.D. 600 value of 2.00.

Bacteria 52 Mn Uptake
Radioactive 52 Mn 2+ (t1 /2 = 5.59 d) decays 29.6% by positron emission. Positron annihilation resulting in two coincident gamma rays at 511 keV energy makes this radioisotope ideal for tracking plant uptake of manganese via gamma counting. For these studies, we purchased a 1 mCi dose of 52 Mn 2+ (equivalent to 37 MBq) from the University of Wisconsin [34]. Details on how this isotope was produced are found in the Supplemental Methods. Upon receipt of the radioisotope, the dose was pH neutralized since it was shipped in dilute HCl and it was diluted further with HyPure™ sterile deionized water for administration to liquid cultures of bacteria isolates following the same procedures described for the 59 Fe studies. Samples were incubated on a rocking platform for 1, 3, and 5 h time points either under LED lights or wrapped in aluminum foil for dark studies.

Statistical Analysis
Data were subjected to one-way analysis of variance (ANOVA) in R using SigmaPlot 14.5. Tukey's HSD test was used for post hoc correction of comparisons across the treatment conditions (i.e., light vs. dark and red vs. blue light) at a significance level of p < 0.05. The 59 Fe 3+/2+ and 52 Mn 2+ allocation data were also analyzed by Principal Component Analysis (PCA) using XLSTAT software version 2020.3 (Addinsoft Inc., New York, NY 10001, USA).

Light Dependencies of BNF and Its Supporting Processes in A. brasilense
To investigate whether the activity level of the bacterial nitrogenase enzyme, responsible for BNF capacity, exhibits a light dependency, an ARA was performed with mutant bacteria inoculated maize roots in both full spectrum white light and darkness conditions ( Figure 1A-C), as well as in red light and blue light conditions ( Figure 1D,E). Regardless of light vs. dark conditions, the ethylene levels slowly increased over the longer root incubation time. This is expected based on natural root emissions of their own ethylene while incubating in the closed jar, as is also measured on non-inoculated maize roots [19]. Natural root ethylene emissions were more obvious with FP10, a non-BNF strain where the slow rise in ethylene over time was due to this process. ARA data were normalized for the total root mass in each sample and average microbial content determined from drop plate assays. However, we did not correct for the ethylene contributions derived from natural root emissions because they were miniscule [19] relative to the levels of ethylene generated by the ARA and did not confound interpretation of microbial light dependencies. the slow rise in ethylene over time was due to this process. ARA data were normalized for the total root mass in each sample and average microbial content determined from drop plate assays. However, we did not correct for the ethylene contributions derived from natural root emissions because they were miniscule [19] relative to the levels of ethylene generated by the ARA and did not confound interpretation of microbial light dependencies. It is clear from the light/dark ARA studies that the BNF-capable bacteria mutants, HM053 and ipdC, showed a significant light dependency in terms of ethylene production indicating that the nitrogenase enzyme in both these strains was more active in the presence of light than in darkness. HM053, which fixes much more nitrogen than ipdC, had values much greater than those observed in the latter especially while exposed to light, producing upwards of 1000 µmol ethylene per gram fresh weight of root (µmol ethylene gfw −1 ) after 6 h compared to the 90 µmol ethylene gfw −1 , respectively. Strikingly, the FP10 mutant, which does not contain an active nitrogenase gene, did not exhibit a light dependency. This seems to point to light sensitivity of the nitrogenase enzyme, or at least the BNF capacity of the bacteria, in A. brasilense.
Following the white light studies, comparisons were made between red (660 nm) and blue light (445 nm) stimulation of nitrogenase activity in BNF-capable mutants of HM053 and ipdC. Here, it was noted that HM053 and ipdC both favored red light for stimulating their nitrogenase enzymes. Additionally, while the wavelength specific light studies were performed at higher light intensities (i.e., 4 times that of the full spectrum white light source) the nitrogenase enzyme in HM053 showed a 6-fold higher level of activity in red light than in the full-spectrum white light after correcting for this intensity difference. On the other hand, blue light stimulation showed approximately the same level of enzyme activity as the full spectrum white light after intensity adjustments were made. Applying the same adjustments for differences in light intensity to the ipdC bacteria, we found that It is clear from the light/dark ARA studies that the BNF-capable bacteria mutants, HM053 and ipdC, showed a significant light dependency in terms of ethylene production indicating that the nitrogenase enzyme in both these strains was more active in the presence of light than in darkness. HM053, which fixes much more nitrogen than ipdC, had values much greater than those observed in the latter especially while exposed to light, producing upwards of 1000 µmol ethylene per gram fresh weight of root (µmol ethylene gfw −1 ) after tions were observed in the FeMo protein allowing the enzyme to reduce CO 2 to CH 4 [36]. In these bacteria, the nitrogenase-variant enzyme was seen to produce methane only in the presence of light and more methane as the light intensity increased with the limitation that the highest intensity explored was 60 µmol m −2 s −1 [35]. While light influences on nitrogenase function in non-photosynthetic bacteria have not been explored; light clearly is shown in these instances to influence the BNF capacity of these organisms. A. brasilense functional mutants did not exhibit BNF inhibition with exposure to light, as noted for Crocosphaera watsonii, suggesting that light influences on BNF activity are perhaps not conserved between photosynthetic and non-photosynthetic bacteria species.
Our interest in ATP biosynthesis as a supporting process is due to BNF being a multielectron redox process carried out by the nitrogenase enzyme in all diazotrophic species which serves to reduce atmospheric nitrogen to a biologically usable form, NH 3 [37], and is driven by ATP as its chemical energy source. BNF requires at least 16 Mg-ATP molecules to reduce a single dinitrogen molecule [38]. Because nitrogen fixation is so energetically costly, its regulation is essential for balancing BNF with growth performance of any diazotrophic microorganism. Nitrogenase activity is regulated transcriptionally through NifA, the transcriptional activator [9], and post-translationally involves dinitrogenase reductaseactivating glycohydrolase together with the P II protein GlnZ [39][40][41].
Many phototrophic microorganisms such as algae and cyanobacteria carry out photophosphorylation, a light dependent process involving the phosphorylation of ADP making ATP. However, to the best of our knowledge, nothing is known regarding nonphototrophic microorganisms such as A. brasilense and whether its ability to biosynthesize ATP is light-dependent.
Comparative measurements using the luciferase chemiluminescence assay were conducted in red and blue light conditions using the HM053, ipdC, and FP10 mutant strains ( Figure 2). All three strains exhibited strong light dependencies in their cellular ATP concentration for both red and blue light when compared to darkness, although in all cases, there was a strong preference for blue light. Although A. brasilense is non-phototrophic, this observation is consistent with past work demonstrating that blue light was most efficient at promoting photophosphorylation in phototrophic microorganisms [38]. red light stimulation boosted nitrogenase activity 17-fold relative to full-spectrum white light, while blue light stimulation resulted in approximately the same level of enzyme activity as the full-spectrum white light.
Light sensitivity in the nitrogenase function has been observed in oceanic photosynthetic bacteria, Crocosphaera watsonii, which, upon exposure of the bacteria to blue light, caused a decrease in nitrogenase activity [35]. In another photosynthetic bacteria species, Rhodopseudomonas palustris, a nitrogenase variant exists in which two amino acid substitutions were observed in the FeMo protein allowing the enzyme to reduce CO2 to CH4 [36]. In these bacteria, the nitrogenase-variant enzyme was seen to produce methane only in the presence of light and more methane as the light intensity increased with the limitation that the highest intensity explored was 60 µmol m −2 s −1 [35]. While light influences on nitrogenase function in non-photosynthetic bacteria have not been explored; light clearly is shown in these instances to influence the BNF capacity of these organisms. A. brasilense functional mutants did not exhibit BNF inhibition with exposure to light, as noted for Crocosphaera watsonii, suggesting that light influences on BNF activity are perhaps not conserved between photosynthetic and non-photosynthetic bacteria species.
Our interest in ATP biosynthesis as a supporting process is due to BNF being a multielectron redox process carried out by the nitrogenase enzyme in all diazotrophic species which serves to reduce atmospheric nitrogen to a biologically usable form, NH3 [37], and is driven by ATP as its chemical energy source. BNF requires at least 16 Mg-ATP molecules to reduce a single dinitrogen molecule [38]. Because nitrogen fixation is so energetically costly, its regulation is essential for balancing BNF with growth performance of any diazotrophic microorganism. Nitrogenase activity is regulated transcriptionally through NifA, the transcriptional activator [9], and post-translationally involves dinitrogenase reductase-activating glycohydrolase together with the PII protein GlnZ [39][40][41].
Many phototrophic microorganisms such as algae and cyanobacteria carry out photophosphorylation, a light dependent process involving the phosphorylation of ADP making ATP. However, to the best of our knowledge, nothing is known regarding non-phototrophic microorganisms such as A. brasilense and whether its ability to biosynthesize ATP is light-dependent.
Comparative measurements using the luciferase chemiluminescence assay were conducted in red and blue light conditions using the HM053, ipdC, and FP10 mutant strains ( Figure 2). All three strains exhibited strong light dependencies in their cellular ATP concentration for both red and blue light when compared to darkness, although in all cases, there was a strong preference for blue light. Although A. brasilense is non-phototrophic, this observation is consistent with past work demonstrating that blue light was most efficient at promoting photophosphorylation in phototrophic microorganisms [38]. Nitrogenase is a two-protein enzyme where one protein contains an iron center and the other an iron-molybdenum center [42]. Iron protein is the only known electron donor to support BNF, and thus, is vital for its function. The predominant oxidation state of the iron in the nitrogenase iron protein is Fe 2+ [42].
In the present work, we explored whether microbial iron uptake, as Fe 3+ and Fe 2+, exhibited certain light dependencies. Using radioactive 59 Fe in its ferric 59 Fe 3+ oxidation state, or in its ferrous 59 Fe 2+ oxidation state, we were able to measure microbial uptake of tracer over a 5 h incubation period as a function of light versus darkness for HM053, ipdC, and FP10 mutant strains. As noted earlier, the light studies used a large red/blue light panel that illuminated the entire culture tube rocker assembly with red/blue light of equal intensities. Results in Figure 3A-C show the uptake curves for 59 Fe 3+ in the three bacteria strains. Interestingly, HM053 showed a strong light dependency for 59 Fe 3+ uptake where more ferric iron was taken up in darkness than in light. Neither ipdC, nor FP10 showed a light dependency for 59 Fe 3+ uptake. Contrary to this, HM053 and ipdC showed a light dependency for 59 Fe 2+ uptake where ferrous iron uptake was significantly higher in light than in darkness ( Figure 3D,E). This observation correlates well with ARA data in Figure 1, and the fact that Fe 2+ is vital to the function of the nitrogenase enzyme in the HM053 and ipdC strains. However, our HM053 ARA data showed much higher levels of nitrogenase activity than ipdC, but the data in Figure 3 indicate that ipdC acquired more 59 Fe 2+ over the 5 h incubation period than HM053. This suggests that Fe 2+ likely plays other roles in the growth and function of these microorganisms. Consistent, too, with ARA data FP10, the BNF deficient strain did not show a light dependency for 59 Fe 2+ uptake ( Figure 3F).
Upon surveying the early literature that addressed metal nutrient uptake and metabolic transformation within live cells of rhizospheric microorganisms, and particularly Azospirilla, we found the number of published works scarce [43][44][45][46]. It was not until later applications of Mössbauer spectroscopy (reviewed in [47]) that a greater understanding of microbial demands for iron came to light [48][49][50][51][52]. In this collection, studies using wild-type Sp245 A. brasilense grown aeroponically in cultures containing 57 Fe 3+ -nitrilotriacetate complex showed that live cells reduced 33% of the assimilated Fe 3+ -to-Fe 2+ over an 18 h period of growth [48]. Similarly, wild-type Sp7 A. brasilense bacteria reared under the same conditions reduced 22% of the assimilated Fe 3+ -to-Fe 2+ [50]. These Mössbauer studies suggested that cellular Fe 3+ was stored in ferritin-like components which was verified in other work [49,51]. Furthermore, these studies showed that Fe 2+ existed in highly coordinated forms [48,50].
Given the state of understanding of microbial iron metabolism from this extensive body of Mössbauer literature, we wanted to examine whether light affected the metabolic transformation of ferric iron once the metal was taken up by the bacteria cell ( Figure 3G). Consistent with the body of Mössbauer data cited above, our radiotracer results showed that under illumination, all three mutant strains of A. brasilense converted a small but consistent 30% of the 59 Fe 3+ taken up by the bacteria cells to 59 Fe 2+ over the course of the 5 h incubation period. In contrast, when all three mutant strains were kept in darkness, we initially observed a lower level of metabolic transformation of 59 Fe 3+ -to-59 Fe 2+ which amounted to only 15% transformation. However, this level rose sharply to 60% 59 Fe 3+ -to-59 Fe 2+ transformation over the 5 h incubation.
A very different story unfolds when 59 Fe 2+ was administered ( Figure 3H). Here, we observed a small but relatively steady 5% transformation of 59 Fe 2+ -to-59 Fe 3+ when HM053 or the ipdC strains were illuminated, suggesting that their active nitrogenase involved in BNF may rapidly appropriate the assimilated 59 Fe 2+ in the iron protein of the enzyme preventing its conversion. When placed in darkness, we found nitrogenase was substantially downregulated as determined by ARA. Under these conditions, we expect less 59 Fe 2+ to bind to the iron protein and thus more of it remains free to convert to 59 Fe 3+ . In fact, at the shortest 30 min timepoint, we observed 70% transformation of 59 Fe 2+ -to-59 Fe 3+ for HM053 and ipdC strains in darkness. Supporting this theory, studies using FP10, the BNF deficient mutant, showed a consistent level of 75% transformation of 59 Fe 2+ -to-59 Fe 3+ over time under illumination. Like the systematic trend observed in darkness when 59 Fe 3+ tracer was administered, there was a linear change in 59 Fe 2+ -to-59 Fe 3+ transformation with time when 59 Fe 2+ was administered to bacteria which decreased from 70% to 50% transformation over the 5 h incubation period. We note that regardless of the initial oxidation state of the 59 Fe radiotracer administered, when in darkness, all bacteria strains appeared to have a larger portion of cellular 59 Fe 2+ over time.
preventing its conversion. When placed in darkness, we found nitrogenase was substantially downregulated as determined by ARA. Under these conditions, we expect less 59 Fe 2+ to bind to the iron protein and thus more of it remains free to convert to 59 Fe 3+ . In fact, at the shortest 30 min timepoint, we observed 70% transformation of 59 Fe 2+ -to-59 Fe 3+ for HM053 and ipdC strains in darkness. Supporting this theory, studies using FP10, the BNF deficient mutant, showed a consistent level of 75% transformation of 59 Fe 2+ -to-59 Fe 3+ over time under illumination. Like the systematic trend observed in darkness when 59 Fe 3+ tracer was administered, there was a linear change in 59 Fe 2+ -to-59 Fe 3+ transformation with time when 59 Fe 2+ was administered to bacteria which decreased from 70% to 50% transformation over the 5 h incubation period. We note that regardless of the initial oxidation state of the 59 Fe radiotracer administered, when in darkness, all bacteria strains appeared to have a larger portion of cellular 59 Fe 2+ over time.

Light Dependencies of Auxin Biosynthesis and Its Supporting Processes in A. brasilense
One of the best attributes of A. brasilense as an agricultural inoculant lies in its ability to biosynthesize the plant relevant hormone auxin [16,19]. Auxin is best known for its diverse roles in regulating developmental and cellular processes of higher plants impacting axis formation and patterning during post-embryogenesis, vascular elongation, leaf expansion, inflorescence, tropism, and apical dominance [53]. It is also especially important in regulating root development [54,55], where it can cause extensive lateral root patterning and root hair formation. Because of its diverse nature in regulating host growth and development, light dependency on auxin biosynthesis by the A. brasilense microorganism was of interest. Using a spectrophotometric assay, we were able to quantify both the total auxin content contained within the bacteria cells and their liquid growth culture ( Figure 4A), as well as isolate the cellular and liquid culture components to calculate cellular auxin excretion as a relative percentage of the total content ( Figure 4B). depict how the original oxidation state of the 59 Fe tracer was metabolically transformed to its other oxidation state over time after being taken up by the bacteria. Data in Panel G represent the percent metabolic transformation of 59 Fe 3+ -to-59 Fe 2+ .after administration of 59 Fe 3+ . Data in Panel H represent the percent metabolic transformation of 59 Fe 2+ -to-59 Fe 3+ after administration of 59 Fe 2+ .

Light Dependencies of Auxin Biosynthesis and Its Supporting Processes in A. brasilense
One of the best attributes of A. brasilense as an agricultural inoculant lies in its ability to biosynthesize the plant relevant hormone auxin [16,19]. Auxin is best known for its diverse roles in regulating developmental and cellular processes of higher plants impacting axis formation and patterning during post-embryogenesis, vascular elongation, leaf expansion, inflorescence, tropism, and apical dominance [53]. It is also especially important in regulating root development [54,55], where it can cause extensive lateral root patterning and root hair formation. Because of its diverse nature in regulating host growth and development, light dependency on auxin biosynthesis by the A. brasilense microorganism was of interest. Using a spectrophotometric assay, we were able to quantify both the total auxin content contained within the bacteria cells and their liquid growth culture ( Figure  4A), as well as isolate the cellular and liquid culture components to calculate cellular auxin excretion as a relative percentage of the total content ( Figure 4B). Like the 59 Fe radiotracer uptake studies, we utilized the same large high intensity red/blue LED light panel to illuminate the liquid culture vials inside the shaking incubator. Our results show that HM053 and FP10 bacteria strains exhibit a significant light dependency when comparing light versus dark condition values. In previous studies, both strains have been reported to biosynthesize auxin at rates of 13.4 ± 0.9 molecules s −1 cell −1 and 7.0 ± 0.4 molecules s −1 cell −1 , respectively, as determined by our direct radiotracer assay [19], while the ipdC auxin deficient mutant strain was estimated to be approximately 10% that of the lower rate. Consistent with earlier findings, the ipdC strain did not show a light dependency when comparing the total auxin content between light and dark conditions. Likewise, the relative percent cellular excretion of auxin was the same across all bacterial strains of A. brasilense examined and did not appear to change when measurements were conducted in light versus darkness. Thus, we can conclude that the process of microbial Like the 59 Fe radiotracer uptake studies, we utilized the same large high intensity red/blue LED light panel to illuminate the liquid culture vials inside the shaking incubator. Our results show that HM053 and FP10 bacteria strains exhibit a significant light dependency when comparing light versus dark condition values. In previous studies, both strains have been reported to biosynthesize auxin at rates of 13.4 ± 0.9 molecules s −1 cell −1 and 7.0 ± 0.4 molecules s −1 cell −1 , respectively, as determined by our direct radiotracer assay [19], while the ipdC auxin deficient mutant strain was estimated to be approximately 10% that of the lower rate. Consistent with earlier findings, the ipdC strain did not show a light dependency when comparing the total auxin content between light and dark conditions. Likewise, the relative percent cellular excretion of auxin was the same across all bacterial strains of A. brasilense examined and did not appear to change when measurements were conducted in light versus darkness. Thus, we can conclude that the process of microbial cellular auxin excretion is diffusional and likely driven by differences between the physical/chemical properties of the inner cellular and extracellular matrices.
Tryptophan is a key aromatic amino acid precursor in auxin biosynthesis and derives from the shikimate pathway. The shikimate pathway consists of seven sequential enzymatic steps and begins with an aldol-type condensation of two phosphorylated active compounds, the phosphoenolpyruvic acid (PEP), from the glycolytic pathway, and the carbohydrate D-erythrose-4-phosphate, from the pentose phosphate cycle, to give 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) ( Figure 5). cellular auxin excretion is diffusional and likely driven by differences between the physical/chemical properties of the inner cellular and extracellular matrices.
Tryptophan is a key aromatic amino acid precursor in auxin biosynthesis and derives from the shikimate pathway. The shikimate pathway consists of seven sequential enzymatic steps and begins with an aldol-type condensation of two phosphorylated active compounds, the phosphoenolpyruvic acid (PEP), from the glycolytic pathway, and the carbohydrate D-erythrose-4-phosphate, from the pentose phosphate cycle, to give 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) ( Figure 5). The seven enzymes that catalyze the pathway are known: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS; EC 4.  [56,57]. Hence, a high microbial auxin-producing capacity could lend itself to higher turnover of the shikimate pathway with commensurate higher demands for Mn 2+ and even higher demands for ATP. The latter comes into play in the fifth step of the  Three of the seven enzymatic steps within the shikimate pathway rely on Mn 2+ as a cofactor [56,57]. Hence, a high microbial auxin-producing capacity could lend itself to higher turnover of the shikimate pathway with commensurate higher demands for Mn 2+ and even higher demands for ATP. The latter comes into play in the fifth step of the shikimate pathway. Here, the shikimate kinase enzyme catalyzes the phosphorylation of the shikimic acid by ATP producing shikimic acid 3-phosphate and ADP. We have already shown that microbial ATP biosynthesis in A. brasilense was light-dependent and was up-regulated across the red and blue light spectrum but favored blue light conditions. Here, we examined whether the light dependency observed for auxin biosynthesis would manifest in a similar light dependency in microbial Mn 2+ uptake. Using radioactive 52 Mn 2+ we were able to measure bacterial uptake of this micronutrient over time as a function of exposure to light versus darkness using the same functional mutants of A. brasilense: HM053, ipdC, and FP10 ( Figure 6A-C).
Microorganisms 2023, 11, 1727 12 of 16 shikimate pathway. Here, the shikimate kinase enzyme catalyzes the phosphorylation of the shikimic acid by ATP producing shikimic acid 3-phosphate and ADP. We have already shown that microbial ATP biosynthesis in A. brasilense was light-dependent and was upregulated across the red and blue light spectrum but favored blue light conditions. Here, we examined whether the light dependency observed for auxin biosynthesis would manifest in a similar light dependency in microbial Mn 2+ uptake. Using radioactive 52 Mn 2+ we were able to measure bacterial uptake of this micronutrient over time as a function of exposure to light versus darkness using the same functional mutants of A. brasilense: HM053, ipdC, and FP10 ( Figure 6 A-C). Surprisingly, the two high auxin-producing mutants HM053 and FP10 did not show the expected light dependency in their ability to assimilate 52 Mn 2+ . Consistent with a diffusional micronutrient exchange between the growth matrix and the microbial cell, both mutants showed increased uptake of radiotracer over time. However, FP10, which has a lower rate of auxin biosynthesis than HM053 [19] consistently assimilated more 52 Mn 2+ than HM053 over the different incubations timepoints examined. Even more surprisingly, the ipdC auxin deficient mutant did exhibit a strong light dependency in its ability to assimilate 52 Mn 2+ and assimilated much more 52 Mn 2+ in darkness than in light. Altogether, these results strongly suggest that microbial Mn 2+ demands are not directly aligned with their auxin-producing capacity as originally hypothesized. Of course, the role of Mn 2+ in microbial function likely translates beyond what we described for the shikimate pathway and auxin biosynthesis. More studies are needed to better understand its roles in microbial biology.
Considering that there were interesting light dependencies for 59 Fe 3+ , 59 Fe 2+ , and 52 Mn 2+ across the different functional mutants of A. brasilense, it was of interest to subject the data to Principal Component Analysis across treatment types to elucidate trends (Figure 7). Here, the information included in our uptake measurements were represented by feature vectors (F1 and F2) representing 56.76% and 43.24% of the information embedded in the 59 Fe 3+ and 59 Fe 2+ data comparison ( Figure 6A), and 78.29% and 21.71% of the information embedded in the 59 Fe 2+ and 52 Mn 2+ data comparison ( Figure 6B). Data points from each of the treatment conditions clustered together, indicating behavior within a treatment type that was similar and distinct from other treatment types. Of greatest note, HM053 mutants in the dark showed elevated uptake of ferric and ferrous radiotracer, indicated by the clustering apart from all other treatments and location toward the end of the ferric feature vector and overall positive Y-value. The auxin-deficient mutant, ipdC, also showed greatest ferrous radiotracer uptake but this was not observed until exposed to light; otherwise, the ipdC mutant clustered near to the FP10 indicating not much difference, other than a slightly elevated ferric uptake, in iron uptake capacity in the dark Surprisingly, the two high auxin-producing mutants HM053 and FP10 did not show the expected light dependency in their ability to assimilate 52 Mn 2+ . Consistent with a diffusional micronutrient exchange between the growth matrix and the microbial cell, both mutants showed increased uptake of radiotracer over time. However, FP10, which has a lower rate of auxin biosynthesis than HM053 [19] consistently assimilated more 52 Mn 2+ than HM053 over the different incubations timepoints examined. Even more surprisingly, the ipdC auxin deficient mutant did exhibit a strong light dependency in its ability to assimilate 52 Mn 2+ and assimilated much more 52 Mn 2+ in darkness than in light. Altogether, these results strongly suggest that microbial Mn 2+ demands are not directly aligned with their auxin-producing capacity as originally hypothesized. Of course, the role of Mn 2+ in microbial function likely translates beyond what we described for the shikimate pathway and auxin biosynthesis. More studies are needed to better understand its roles in microbial biology.
Considering that there were interesting light dependencies for 59 Fe 3+ , 59 Fe 2+ , and 52 Mn 2+ across the different functional mutants of A. brasilense, it was of interest to subject the data to Principal Component Analysis across treatment types to elucidate trends ( Figure 7). Here, the information included in our uptake measurements were represented by feature vectors (F1 and F2) representing 56.76% and 43.24% of the information embedded in the 59 Fe 3+ and 59 Fe 2+ data comparison ( Figure 6A), and 78.29% and 21.71% of the information embedded in the 59 Fe 2+ and 52 Mn 2+ data comparison ( Figure 6B). Data points from each of the treatment conditions clustered together, indicating behavior within a treatment type that was similar and distinct from other treatment types. Of greatest note, HM053 mutants in the dark showed elevated uptake of ferric and ferrous radiotracer, indicated by the clustering apart from all other treatments and location toward the end of the ferric feature vector and overall positive Y-value. The auxin-deficient mutant, ipdC, also showed greatest ferrous radiotracer uptake but this was not observed until exposed to light; otherwise, the ipdC mutant clustered near to the FP10 indicating not much difference, other than a slightly elevated ferric uptake, in iron uptake capacity in the dark relative to FP10 in either light condition. The mutant FP10 in both light and dark were nearly indistinguishable from one another and thus, did not change under light stimulus. This demonstrated nicely that the 59 Fe uptake capacity of the bacteria correlated with BNF capacity of the mutant strain while also showing a light dependency when BNF capacity was available to the bacteria. Additionally, FP10 showed the most similar behavior in comparing ferrous iron and manganese uptake, while ipdC showed the most dissimilarity here owing to its significant light dependency noted for 52 Mn 2+ uptake.

Conclusions
As the increased implementation of bacterial inoculants for the enhancement of agricultural crop yield and plant nutrition continues, it becomes more important to fully understand the biological functions and mechanisms of action behind their usefulness. Here, we investigated the light dependencies for two important microbial functions associated with plant growth promotion including BNF and auxin biosynthesis. Although not a phototrophic organism, we discovered that both these functions were light sensitive in A. brasilense, being upregulated in light as opposed to darkness. Additionally, we discovered that certain supporting functions underpinning BNF, and auxin biosynthesis, were also light sensitive. Here, we found that microbial uptake of Fe 2+ as a key element in forming the nitrogen iron protein was light-sensitive, as was ATP, the major energy source for driving BNF and auxin biosynthesis. Consistent with past studies [1], we found that all three strains of A. brasilense showed the same growth performance with each other, and no apparent light dependency. Hence, cellular ATP levels and microbial growth were not directly related. This observation is consistent with past studies in E. coli which showed that bacterial growth rates were independent of their cellular ATP concentration [58].
While the present work was not able to connect observed light dependencies of A. brasilense with its host's ability to conduct light within the roots, we must wonder why these microorganisms have evolved with traits allowing them to respond to light stimuli. In a time when use of microbial inoculants in agriculture to promote plant growth is becoming a commonplace practice, ways to improve a plant's microbiota might seek to boost plant light transmission belowground or seek to improve the light sensing capabilities of these microorganisms.

Conclusions
As the increased implementation of bacterial inoculants for the enhancement of agricultural crop yield and plant nutrition continues, it becomes more important to fully understand the biological functions and mechanisms of action behind their usefulness. Here, we investigated the light dependencies for two important microbial functions associated with plant growth promotion including BNF and auxin biosynthesis. Although not a phototrophic organism, we discovered that both these functions were light sensitive in A. brasilense, being upregulated in light as opposed to darkness. Additionally, we discovered that certain supporting functions underpinning BNF, and auxin biosynthesis, were also light sensitive. Here, we found that microbial uptake of Fe 2+ as a key element in forming the nitrogen iron protein was light-sensitive, as was ATP, the major energy source for driving BNF and auxin biosynthesis. Consistent with past studies [1], we found that all three strains of A. brasilense showed the same growth performance with each other, and no apparent light dependency. Hence, cellular ATP levels and microbial growth were not directly related. This observation is consistent with past studies in E. coli which showed that bacterial growth rates were independent of their cellular ATP concentration [58].
While the present work was not able to connect observed light dependencies of A. brasilense with its host's ability to conduct light within the roots, we must wonder why these microorganisms have evolved with traits allowing them to respond to light stimuli. In a time when use of microbial inoculants in agriculture to promote plant growth is becoming a commonplace practice, ways to improve a plant's microbiota might seek to boost plant light transmission belowground or seek to improve the light sensing capabilities of these microorganisms.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms11071727/s1, and includes Supplemental Methods and the following Supplemental Figures: Figure S1, Assembly for preparing batches of acetylene gas for the Acetylene Reduction Assay; Figure S2, Sample collection jar containing maize roots inoculated with A. brasilense bacteria; Figure S3, Ion chromatography station used to separate microbial 59 Fe 3+ and 59 Fe 2+ after radiotracer uptake; Figure S4, Chemiluminescence spots from the Luciferase Assay using the Typhoon 9000 Imager showing ATP standards and samples done in triplicate for HM053, ipdC, and FP10 strains of A. brasilense bacteria; and Figure S5, Optical cuvettes showing color differences between HM053, a high auxin producing strain, and ipdC, an auxin deficient strain of A. brasilense bacteria.